Priming effects of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 on the development of adenomyosis in the mouse uterus

The possibility of therapeutic application of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 for preventing the development of uterine adenomyosis was investigated in mice. First priming effects of CP8816 on 17beta-estradiol (E2)-induced cell division in uterine tissues were examined. As a result, pretreatment with CP8816 or progesterone significantly suppressed the elevation of the mitotic activity in the luminal epithelial cells of mice treated with E2 later. Priming with CP8816 had little effect on the stromal cells, but progesterone priming caused an increase of stromal mitotic activity in mice treated with E2 later. To evaluate the inhibitory effect of these compounds on the development of adenomyosis induced experimentally by pituitary grafting, 7-week-old female mice were isografted with a single anterior pituitary in the uterus and divided into four groups. Two groups of mice were given daily subcutaneous injections of 1 mg of CP8816 or the vehicle alone for 6 weeks from the day after the grafting. Remaining two groups of mice were given oral administration of 1 mg of CP8863 or the vehicle only for 5 weeks starting one week after the grafting. The incidence of adenomyosis was significantly lower in the groups of mice treated with CP8816 and CP8863 than in the respective control groups. The mechanism by which CP compounds inhibited the development of adenomyosis might be related to their priming effects, i.e., their inhibitory effect on epithelial cell division and lack of effect on stromal cell division after subsequent exposure to E2.     

Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.     

Expression of programmed death 1 ligands by murine T cells and APC

Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-gamma, GM-CSF, or IL-4, and on DCs by IFN-gamma, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-gamma, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.     

Cutting edge: tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-alpha-induced suppression of B lymphocyte formation

IFN-alpha inhibits B lymphocyte development, and the nuclear protein Daxx has been reported to be essential for this biological activity. We show in this study that IFN-alpha inhibits the clonal proliferation of B lymphocyte progenitors in response to IL-7 in wild-type, but not in tyk2-deficient, mice. In addition, the IFN-alpha-induced up-regulation and nuclear translocation of Daxx are completely abrogated in the absence of tyk2. Therefore, tyk2 is directly involved in IFN-alpha signaling for the induction and translocation of Daxx, which may result in B lymphocyte growth arrest and/or apoptosis.     

Action of xyloglucan hydrolase within the native cell wall architecture and its effect on cell wall extensibility in azuki bean epicotyls

Xyloglucan hydrolase (XGH) has recently been purified from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls as a new type of xyloglucan-degrading enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154]. In the present study, the effects of XGH on the mechanical properties of the cell wall and on the level and the molecular size of xyloglucans within the native wall architecture were examined in azuki bean epicotyls. When the epidermal tissue strips from the growing regions of azuki bean epicotyls were incubated with XGH, the mechanical extensibility of the cell wall dramatically increased. XGH exogenously applied to cell wall materials (homogenates) or epidermal tissue strips decreased the amount of xyloglucans via the solubilization of the polysaccharides. Also, XGH substantially decreased the molecular mass of xyloglucans in both materials. These results indicate that XGH is capable of hydrolyzing xyloglucans within the native cell wall architecture and thereby increasing the cell wall extensibility in azuki bean epicotyls.     

Chiral synthesis of secondary alcohols using Geotrichum candidum

Chiral synthesis of secondary alcohols of both the (S)- and (R)-enantiomer with extremely high enantioselectivities (up to >99% ee) using a biocatalyst, Geotrichum candidum, is reviewed. Resting cell and dried-cell preparation using acetone were applied to oxidation, reduction, and deracemization reactions. Many methods to improve the reactivity and enantioselectivity of the reactions were developed. For example, additives such as secondary alcohols and hydrophobic resin (Amberlite XAD) were used in nonaqueous reaction media such as organic and supercritical solvents as well as in aqueous ones. As a result, optically pure alcohols of both enantiomers were synthesized on a gram scale.     

Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.     

Measurement of bisphenol A in human urine using liquid chromatography with multi-channel coulometric electrochemical detection

Smith-Lemli-Opitz syndrome (SLOS) patients have increased 7- and 8-dehydrocholesterol (DHC) concentrations. Using gas chromatography-mass spectrometry with selected ion monitoring we investigated whether storage time (24 h, 7 and 30 days, and 22 months at room temperature or at 4 degrees C) affected DHC concentrations in whole blood spots (WBSs) from SLOS patients and normal controls. Our results suggest that WBS sterol analysis can be used for SLOS screening and possibly related inborn errors of sterol metabolism with a 100% sensitivity and specificity on specimens stored for up to 30 days, either at room temperature or 4 degrees C. After 22 months of storage at both temperature SLOS samples can be indistinguishable from control samples. Therefore, great caution should be used to exclude SLOS by sterol analysis of WBSs stored for a long time.